Journal: bioRxiv

Article Title: RNA polymerase II clusters form in line with liquid phase wetting of chromatin

doi: 10.1101/2021.02.03.429626

Figure Lengend Snippet: A) Sketch of the recruitment and release of RNA polymerase II (Pol II) in the context of macromolecular clusters. B) Representative maximum projection of a nucleus in a live zebrafish embryo (sphere stage), where Pol II was detected via fluorescently labeled antibody fragments (Fab) specific against Ser2 and Ser5 phosphorylation (Pol II Ser2P, Pol II Ser5P). Pol II Ser5P clusters representing the different apparent types of morphologies are marked. Single time point z stacks were recorded using an instantaneous Structured Illumination Microscope (instant-SIM) microscope, raw data were processed by local background subtraction. C) Detail views of the clusters of the apparent morphological types i-iii, as marked in panel A. D) Examples of the varied morphologies of type iii clusters, shown as maximum projections and corresponding volume renderings of the processed Pol II Ser5P signal. Morphologies are named by similarity to patisserie and candy items. (3D renderings ImageJ Volume Viewer plugin).

Article Snippet: Primary antibodies against Pol II Ser5P (clone 4H8, abcam ab5408, mouse monoclonal IgG diluted 1:300) and Pol II Ser2P (abcam ab193468, rabbit monoclonal IgG, diluted 1:2500) were applied overnight at 4°C in 4% BSA in PBST.

Techniques: Labeling, Microscopy

Journal: bioRxiv

Article Title: RNA polymerase II clusters form in line with liquid phase wetting of chromatin

doi: 10.1101/2021.02.03.429626

Figure Lengend Snippet: Clusters were visualized in live zebrafish embryos (sphere stage) using antibody fragments (Fab, Cy3-labeled) against the RNA polymerase II carboxyterminal domain serine 5 phosphorylation. The time-lapse images are maximum projections from a data set with 40 s time resolution signal. Images were chosen at selected time points to show short-term and long-term behavior. The same persistence of cluster morphologies was observed in time lapse sequences recorded from three embryos in parallel in the same experiment, and in a second independent repeat of this experiment.

Article Snippet: Primary antibodies against Pol II Ser5P (clone 4H8, abcam ab5408, mouse monoclonal IgG diluted 1:300) and Pol II Ser2P (abcam ab193468, rabbit monoclonal IgG, diluted 1:2500) were applied overnight at 4°C in 4% BSA in PBST.

Techniques: Labeling

Journal: bioRxiv

Article Title: RNA polymerase II clusters form in line with liquid phase wetting of chromatin

doi: 10.1101/2021.02.03.429626

Figure Lengend Snippet: A) Representative nuclear mid-section obtained by STEDD super-resolution microscopy from a fixed late blastula stage zebrafish embryo. Pol II Ser5P intensity distributions were obtained by STEDD microscopy, Pol II Ser2P intensity distributions by regular confocal microscopy from the same focal plane. Pol II Ser5P clusters with typical morphologies i-iii are marked. B) Detail views of the marked clusters, representing the typical morphologies i-iii. C) Area and solidity of individual clusters, with gate regions for the typical morphologies i-iii. Clusters were segmented based on Pol II Ser5P intensity, data obtained from a total of 52 mid-nuclear sections from two different samples. D) The Pol II Ser5P and Pol II Ser2P intensities (mean intensity across all pixels inside a given cluster’s segmentation mass) of the clusters in the gates i-iii are plotted in color over the entire ungated cluster population (light gray). Mean Pol II Ser5P and Pol II Ser2P intensities were scaled by the median value for each nucleus, then pooled. E) The median of the Pol II Ser5P and Pol II Ser2P levels of the gated clusters in panel D is plotted over the ungated population of clusters. Each cluster type is plotted with 95% bootstrapped confidence intervals in Pol II Ser5P and Pol II Ser2P direction (10,000 resamples). F) Analysis of the placement of Pol II Ser2P spots relative to Pol II Ser5P clusters (spots segmented based on Pol II Ser2P channel). Histograms show the distribution of distances to the nearest surface of a Pol II Ser5P cluster (Euclidean metric, applied to individual pixels, negative values are from outside, positive values from inside a Pol II Ser5P cluster). The distance distribution of pixels inside Pol II Ser5P clusters is shown for reference. G) Sketch of apparent morphological types of Pol II Ser5P clusters, placed by their levels of elongating and paused Pol II.

Article Snippet: Primary antibodies against Pol II Ser5P (clone 4H8, abcam ab5408, mouse monoclonal IgG diluted 1:300) and Pol II Ser2P (abcam ab193468, rabbit monoclonal IgG, diluted 1:2500) were applied overnight at 4°C in 4% BSA in PBST.

Techniques: Microscopy, Confocal Microscopy

Journal: bioRxiv

Article Title: RNA polymerase II clusters form in line with liquid phase wetting of chromatin

doi: 10.1101/2021.02.03.429626

Figure Lengend Snippet: Cluster properties were extracted from a zebrafish embryo (sphere stage) during live imaging using antibody fragments (for details, see Materials and Methods). A) Area and solidity of the individual clusters, with gates showing the type i, ii and iii clusters. B) Phosphorylation levels (serine 2 and serine 5 phosphorylation) of the individual clusters were monitored by mean fluorescence intensity. Clusters belonging to one gate tend to have similar Pol II Ser2P and Pol II Ser5P intensities. The values are normalized to the median value. Number of nuclei: n = 109. Number of clusters: n = 959.

Article Snippet: Primary antibodies against Pol II Ser5P (clone 4H8, abcam ab5408, mouse monoclonal IgG diluted 1:300) and Pol II Ser2P (abcam ab193468, rabbit monoclonal IgG, diluted 1:2500) were applied overnight at 4°C in 4% BSA in PBST.

Techniques: Imaging, Fluorescence

Journal: bioRxiv

Article Title: RNA polymerase II clusters form in line with liquid phase wetting of chromatin

doi: 10.1101/2021.02.03.429626

Figure Lengend Snippet: A) Sketch of cluster nucleation with all particle and polymer species involved in the model. B) Interaction matrix for different species in the lattice model. Affinity is represented by negative and repulsion by positive values. C) Example lattice configurations for all three morphological cluster types (i-iii), are shown as lattice simulation output and corresponding synthetic microscopy images. D) Area and solidity of individual clusters, with gate regions for the typical morphologies i-iii. Clusters were segmented based on Pol II Ser5P intensity (total 20401 clusters). The simulated configurations contained chains with different numbers of gray ( N AC = 0,3,6), blue ( N PC = 2,4,6,8) and black ( N IC depending on N AC and N PC ) monomers, with a total polymer length L polymer = 20. 6525 clusters in gate i, 4065 clusters in gate ii, 2326 clusters in gate iii. E) The Pol II Ser5P and Pol II Ser2P intensities (mean intensity across all pixels inside a given cluster’s segmentation mass) of the clusters in the gates i-iii are plotted in color over the entire ungated cluster population (light gray). F) The median of the Pol II Ser5P and Pol II Ser2P levels of the gated clusters in panel D is plotted over the ungated population of clusters. Each cluster type is plotted with 95% bootstrapped confidence intervals in Pol II Ser5P and Pol II Ser2P direction (10,000 resamples). For types i and iii, the confidence interval is hidden by the median data point. Mean Pol II Ser5P and Pol II Ser2P intensities were scaled by the median value for all time steps.

Article Snippet: Primary antibodies against Pol II Ser5P (clone 4H8, abcam ab5408, mouse monoclonal IgG diluted 1:300) and Pol II Ser2P (abcam ab193468, rabbit monoclonal IgG, diluted 1:2500) were applied overnight at 4°C in 4% BSA in PBST.

Techniques: Microscopy

Journal: bioRxiv

Article Title: RNA polymerase II clusters form in line with liquid phase wetting of chromatin

doi: 10.1101/2021.02.03.429626

Figure Lengend Snippet: A) RNA polymerase II Ser5 and Ser2 phosphorylation throughout the nucleus as obtained by immunofluorescence from primary cell cultures of zebrafish embryos, treated with the transcription inhibitors triptolide (TL), flavopiridol (FL), and actinomycin D (ActD). Intensity levels are shown as quantile plots (state quantiles), intensities are mean values after subtraction of cytoplasmic background, which can result in negative values. Within a given experimental repeat, values are normalized by the median of the control condition. Intensities are given as median with boxplots. The number of distinct clusters per nucleus is shown as mean±SEM. *** indicates p < 0.001, ** indicates p < 0.01, * indicates p < 0.05, n.s. indicates p ≥ 0.05 (Int. Pol II Ser5P: p = 0.04, p = 0.05, p < 0.0001, Int. Pol II Ser2P: p = 0.0002, p < 0.0001, p < 0.0001, Number of clusters: p = 0.004, p = 0.0002, p = 0.0006 for differences from the control condition, Bonferroni correction for multiple testing, n = 165,118,147,107 nuclei per condition from three independent experimental repeats.) B) Corresponding statistics for cultured THP-1 cells. (Int. Pol II Ser5P: p = 0.003, p = 0.61, p = 0.008, Int. Pol II Ser2P: p < 0.0001, p = 0.30, p = 0.009, Number of clusters: p = 0.10, p = 1.08, p = 0.10 for differences from the control condition, Bonferroni correction for multiple testing, n = 158,79,93,87 nuclei from three independent experimental repeats.)

Article Snippet: Primary antibodies against Pol II Ser5P (clone 4H8, abcam ab5408, mouse monoclonal IgG diluted 1:300) and Pol II Ser2P (abcam ab193468, rabbit monoclonal IgG, diluted 1:2500) were applied overnight at 4°C in 4% BSA in PBST.

Techniques: Immunofluorescence, Cell Culture

Journal: bioRxiv

Article Title: RNA polymerase II clusters form in line with liquid phase wetting of chromatin

doi: 10.1101/2021.02.03.429626

Figure Lengend Snippet: A) Pol II Ser5P and Pol II Ser2P mean intensity, area, and solidity of individual clusters in primary cell cultures established from oblong stage zebrafish embryos. Cultures were incubated for 30 minutes with inhibitors as indicated. Cluster intensities are mean intensities calculated over all pixels of maximum-projected clusters, cytoplasmic staining background was subtracted, and values were normalized by the median intensity of the control samples of a given experimental repeat (indicated by lines at intensity level 1). Shown are median values with boxplots. *** indicates p < 0.001, ** indicates p < 0.01, * indicates p < 0.05, n.s. indicates p ≥ 0.05 (Int. Pol II Ser5P: p < 0.0001, p = 0.25, p = 0.10, Int. Pol II Ser2P: p < 0.0001, p < 0.0001, p < 0.0001, Area: p = 0.26, p = 0.06, p = 2.74, Solidity: p = 0.0007, p < 0.0001, p = 0.001 for differences from the control condition, Bonferroni correction for multiple testing, n = 1514,1631,703,1567 clusters per condition from three independent experimental repeats.) B) Corresponding properties of clusters in undifferentiated THP-1 cells treated exactly like zebrafish primary cell culture. (Int. Pol II Ser5P: p < 0.0001, p = 0.36, p = 1.90, Int. Pol II Ser2P: p < 0.0001, p < 0.0001, p < 2.01, Area: p = 1.19, p = 0.10, p = 2.78, Solidity: p = 0.0004, p = 0.03, p = 0.01 for differences from the control condition, Bonferroni correction for multiple testing, n = 402,294,150,114 clusters per condition from three independent experimental repeats.)

Article Snippet: Primary antibodies against Pol II Ser5P (clone 4H8, abcam ab5408, mouse monoclonal IgG diluted 1:300) and Pol II Ser2P (abcam ab193468, rabbit monoclonal IgG, diluted 1:2500) were applied overnight at 4°C in 4% BSA in PBST.

Techniques: Incubation, Staining, Cell Culture

Journal: bioRxiv

Article Title: RNA polymerase II clusters form in line with liquid phase wetting of chromatin

doi: 10.1101/2021.02.03.429626

Figure Lengend Snippet: A) Mean Pol II Ser5P and Pol II Ser2P intensity distribution of Pol II clusters in data recorded from primary zebrafish cell cultures treated with DMSO control media (Ctrl), triptolide (TL), or flavopiridol (FP) for 30 minutes. B) Sketch of the mathematical model that was applied to reproduce changes in Pol II Ser5P and Pol II Ser2P intensities. A droplet containing Pol II Ser5P is the central model compartment, to which Pol II Ser5P is recruited (rate coefficient k A ), and released either via the compartment boundary (rate coefficient kp ) or by transition into transcription elongation (rate coefficient k R ). C) Parameter changes used to reproduce the intensity distribution in the inhibitor-treated samples. D) Intensity distributions were reproduced based on the mathematical model and the control-treated intensity distribution. For every cluster in the control condition, the parameter values ( I,C ) were calculated, and subsequently transformed into altered Pol II Ser5P and Pol II Ser2P intensities by multiplication of K A and ρ with a prefactor representing the inhibitor treatment. After triptolide treatment, K A and p were 0.72 and 0.58 fold their control values, respectively. After flavopiridol treatment, K A and p were 1.18 and 0.76 fold their control values, respectively. The parameter estimation was based on minimizing the root-mean-square difference between predicted and measured Pol II Ser5P and Pol II Ser2P intensity distributions, see .

Article Snippet: Primary antibodies against Pol II Ser5P (clone 4H8, abcam ab5408, mouse monoclonal IgG diluted 1:300) and Pol II Ser2P (abcam ab193468, rabbit monoclonal IgG, diluted 1:2500) were applied overnight at 4°C in 4% BSA in PBST.

Techniques: Transformation Assay

Journal: bioRxiv

Article Title: RNA polymerase II clusters form in line with liquid phase wetting of chromatin

doi: 10.1101/2021.02.03.429626

Figure Lengend Snippet: Representative mid-nuclear section nuclei in zebrafish primary cell culture (upper 3 rows) and THP-1 cell line (lower three rows). Cells were treated with control media (Ctrl), triptolide (TL), flavopidirol (FP) or actinomycin D for 30 min, and subsequently stained for Ser2 and Ser5 phosphorylation of RNA polymerase II by indirect immunofluorescence.

Article Snippet: Primary antibodies against Pol II Ser5P (clone 4H8, abcam ab5408, mouse monoclonal IgG diluted 1:300) and Pol II Ser2P (abcam ab193468, rabbit monoclonal IgG, diluted 1:2500) were applied overnight at 4°C in 4% BSA in PBST.

Techniques: Cell Culture, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: RNA polymerase II clusters form in line with liquid phase wetting of chromatin

doi: 10.1101/2021.02.03.429626

Figure Lengend Snippet: A) Histograms of I and p values obtained using Pol II Ser5P and Pol II Ser2P intensity levels of Pol II clusters control-treated primary zebrafish embryo cell cultures. In the calculation of ( Î,ρ), the parameter values K A =0.1 and C =1 were used. These were taken from previous work: K A = k A /k p = 0.1 considers k A ≈ 1 min -1 and k D ≈ 10 min -1 . C = k R Telong ≈ 1 considers k R ≈ 2 min -1 , length of 5 — 10 kb in zebrafish embryos at the late blastula stage of development and an elongation rate of 2 kb/s . To assess the validity of this parameter calculation, the ρ histogram was compared against the expected ρ = k R /k p ≈ 0.05 ( k D ≈ 1/7s, k R ≈ 1/150 s, previously estimated , as indicated by a black line. This comparison suggests strong agreement. B) Fitting of the modified affinity values and ρ ’ for cell cultures treated with DMSO (Control), triptolide, and flavopiridol. Images show the root-mean-square residuals, with white circles indicating the position of the minimal value. Residuals were calculated between the distributions of Pol II Ser5P and Pol II Ser2P intensities obtained from the treatment experiments directly or predicted based on the control condition and a modification of the K A and ρ values. The optimal values of and were chosen based on the location of the minimum.

Article Snippet: Primary antibodies against Pol II Ser5P (clone 4H8, abcam ab5408, mouse monoclonal IgG diluted 1:300) and Pol II Ser2P (abcam ab193468, rabbit monoclonal IgG, diluted 1:2500) were applied overnight at 4°C in 4% BSA in PBST.

Techniques: Modification

Journal: bioRxiv

Article Title: RNA polymerase II clusters form in line with liquid phase wetting of chromatin

doi: 10.1101/2021.02.03.429626

Figure Lengend Snippet: A) Examples of lattice configurations obtained from simulations with parameter changes that represent inhibitor treatment. For the control treatment (Ctrl), a range of Npc = 2,4,6,8 blue monomers per chain and N AC = 0,3,6 gray monomers per chain (total length L polymer = 20) was used; Pol II affinity parameters were assigned standard values ( w Pol-Pol = —0.35, w PC-Pol = —0.5). For the triptolide treatment (TL), the same parameter range for N PC and N AC were used, and Pol II affinities were reduced ( w pol-pol = —0.25, w PC-pol = —0.25). For the flavopiridol treatment (FP), no gray monomers were included ( N AC = 0 throughout), Pol II affinities were unchanged. B) Cluster solidity and number of distinct clusters per simulation. Quantification was based on synthetic microscopy images, Ctrl contained 12,000 evaluated images, TL 12,000, and FP 4,000. Solidity is median with boxplots, number is mean±SEM; *** indicates p < 0.001, * indicates p < 0.05 ( p < 0.0001, and p = 0.03 for solidity, p < 0.0001 and p < 0.0001 for number of clusters; permutation test for differences from control, resampling n matched to experiments in panel C, Bonferroni-corrected). C) Cluster solidity and number of distinct clusters per cell nucleus obtained from primary zebrafish cell cultures. *** indicates p<0.001 ( p = 0.007, and p < 0.0001 with n = 1514,1631,703 clusters for solidity, p = 0.004 and p = 0.0002 with n = 402,294,150 nuclei for number of clusters; permutation test for differences from control, Bonferroni-corrected, data obtained from three independent sets of experiments). Full assessment of cluster and per-nucleus properties, see and .

Article Snippet: Primary antibodies against Pol II Ser5P (clone 4H8, abcam ab5408, mouse monoclonal IgG diluted 1:300) and Pol II Ser2P (abcam ab193468, rabbit monoclonal IgG, diluted 1:2500) were applied overnight at 4°C in 4% BSA in PBST.

Techniques: Microscopy

Journal: bioRxiv

Article Title: RNA polymerase II clusters form in line with liquid phase wetting of chromatin

doi: 10.1101/2021.02.03.429626

Figure Lengend Snippet: A) To test whether our analysis of maximum-projected, two-dimensional (2D) microscopy data is representative of three-dimensional (3D) organization, we compare the number of Pol II Ser5P clusters detected per nucleus in both cases. A plot of the numbers from 2D segmentation based on maximum intensity projection (x-axis) and from 3D segmentation (y-axis) shows only small deviations from the dashed diagonal line (indicating perfect agreement). In the inset, we show the fraction of nuclei in which the number of clusters in the 2D and 3D methods are the same, differ by just 1 cluster, or by 2 and more clusters. The two methods result in a similar number of clusters: 38% of the nuclei have same number, and a total of 72% of nuclei differ in the 2D and 3D clusters at most by 1 cluster. The clusters were segmented using a global Robust Background Threshold (3 standard deviations for 2D projection and 4.5 standard deviations for the 3D image) on the masked images. B) We compare the area and volume of the clusters obtained from the 2D segmentation and 3D segmentation, respectively. For each nucleus, we obtained a set of common clusters in 2D and 3D by comparing their x, y positions (ignoring the z position of the 3D clusters) and picking those that overlap. For these clusters, a direct comparison is possible, and we can see that the volume and area are highly correlated. A linear fit results in a slope of 0.65, which gives an effective mean radius of about 0.5 μ m if the clusters were spherical. Overall, this comparison of 3D and 2D Pol II Ser5P clusters shows that 2D segmentation using maximum intensity projections results in a similar number and size of clusters when compared to a 3D analysis, justifying our approach of using the 2D method for our main analysis.

Article Snippet: Primary antibodies against Pol II Ser5P (clone 4H8, abcam ab5408, mouse monoclonal IgG diluted 1:300) and Pol II Ser2P (abcam ab193468, rabbit monoclonal IgG, diluted 1:2500) were applied overnight at 4°C in 4% BSA in PBST.

Techniques: Microscopy

Journal: bioRxiv

Article Title: RNA polymerase II clusters form in line with liquid phase wetting of chromatin

doi: 10.1101/2021.02.03.429626

Figure Lengend Snippet: A) Algorithm overview. B) Illustration of a possible initial configuration containing a single polymer and all species within the system (black, blue, gray and red). C) Overview table of the interspecies affinity parameters. Table entries represent w values, crosses correspond to no interaction ( w = 0). D) Possible move types for the different species. Pol II can move to all its eight nearest neighbours. Chains representing chromatin can undergo the Verdier-Stockmayer move set. E) Tower-sampling method to choose a transition. F) Minimal area for the local update of possible transitions.

Article Snippet: Primary antibodies against Pol II Ser5P (clone 4H8, abcam ab5408, mouse monoclonal IgG diluted 1:300) and Pol II Ser2P (abcam ab193468, rabbit monoclonal IgG, diluted 1:2500) were applied overnight at 4°C in 4% BSA in PBST.

Techniques: Sampling